Abstract

Using endogenous human cellular ubiquitin system enzymes and added
his-tagged ubiquitin, ATP, and an ATP-regenerating system, we labelled
cellular proteins with hexahistidine tagged ubiquitin in
vitro. Labeling was dependent on ATP and the ATP recycling
system, on the proteasome inhibitor MG132 and the ubiquitin protease
inhibitor ubiquitin aldehyde, and was inhibited by iodoacetamide.
Labeled proteins were affinity extracted in quadruplicate and tryptic
peptides identifed by 2D capillary LC/MS/MS comb9ined with SEQUEST and
MEDUSA analyses. Support vector machine analyais of the mass
spectrometry data allowed prediction of correct matches between mass
spectrometry data and peptide sequences. Overall, 144 proteins were
identified by peptides predicted to be correctly sequenced, and 113
were identified by at least three peptides or one or two peptides with
at least an 80% chance of being correct. Identified proteins included
22 proteasome subunits or associated proteins, 18 E1, E2 or E3
ubiquitin system enzymes or related proteins, and four ubiquitin
domain proteins. Seventeen directly ubiquitinated proteins or
proteins associated with the ubiquitin system were identified.
Functional clusters of other proteins included redox enzymes, proteins
associated with endocytosis, cytoskeletal proteins, DNA damage or
repair related proteins, calcium binding proteins, and splicing factor
and related proteins, suggesting that in vitro ubiquitination
is not random, and that these functions may be regulated by the
ubiquitin system. This map of cellular ubiquitinated proteins and
their interacting proteins will be useful for further studies of
ubiquitin system function.